Project Summary The prevalence of major depressive disorder (MDD), one of the most common disorders in the United States, is doubled in patients with type 2 diabetes mellitus (T2DM). Traditionally thought of as two interacting, though fundamentally independent disorders, emerging evidence suggests that MDD and T2DM may share a common etiology: dysregulated metabolism. Dysregulated metabolism could disrupt neuronal activity in key limbic areas, contributing to MDD. However, little is known about how metabolic processes affect neuronal activity in key limbic areas such as the basolateral amygdala (BLA) in control states, or how these processes become dysregulated under metabolically stressful conditions. The central hypothesis of this study is that AMPK plays an important regulatory role in BLA excitability and affective behavior that becomes dysregulated under metabolically stressful conditions. This project will first elucidate how AMPK activity regulates affective behavior in rats. Using site-specific injection of an AMPK inactivator into the BLA, affective behavior will be tested using the elevated plus maze and forced swim task paradigms. Motor and cognitive behavior will also be tested using the open field maze and novel object recognition task. In addition to pharmacological AMPK inactivation, AAV viruses will be injected into the BLA to express a dominant-negative form of the AMPK ?2 subunit specifically in BLA principal neurons, testing the effect of cell-type specific AMPK inactivation on affective behavior. Secondly, this project will characterize how AMPK activity modulates BLA principal neuron excitability. Using whole-cell patch clamp slice electrophysiology, the effect of intra- and extracellular administration of an AMPK inactivator on membrane resistance, spike threshold, and LTP threshold of BLA principal neurons will be tested. The effects of dominant negative AMPK ?2 subunit expression on these electrophysiological properties will also be measured. Thirdly, the above experiments will be repeated using rats fed a high-fructose diet, a well- characterized model of metabolic stress. We will thus test the effects of the high fructose diet on AMPK activity, affective behavior, and BLA electrophysiology. We will also test if the effects of a high-fructose diet can be rescued through administration of a pharmacological AMPK activator, or cell type specific overexpression of wild-type AMPK ?2. The combination of molecular, electrophysiological, and behavioral techniques will help us understand the link between metabolic state and mood regulation in both normal and dysfunctional states. Elucidating the role of the AMPK signaling cascade in the BLA could contribute to our understanding of MDD etiology and lead to novel antidepressant development.